Platelet-rich plasma alleviates neuropathic pain in osteoarthritis by downregulating microglial activation.

BACKGROUND: The development of neuropathic pain (NP) is one of the reasons why the pain is difficult to treat, and microglial activation plays an important role in NP. Recently, platelet-rich plasma (PRP) has emerged as a novel therapeutic method for knee osteoarthritis (KOA). However, it's unclarified whether PRP has analgesic effects on NP induced by KOA and the underlying mechanisms unknown. PURPOSE: To observe the analgesic effects of PRP on NP induced by KOA and explore the potential mechanisms of PRP in alleviating NP. METHODS: KOA was induced in male rats with intra-articular injections of monosodium iodoacetate (MIA) on day 0. The rats received PRP or NS (normal saline) treatment at days 15, 17, and 19 after modeling. The Von Frey and Hargreaves tests were applied to assess the pain-related behaviors at different time points. After euthanizing the rats with deep anesthesia at days 28 and 42, the corresponding tissues were taken for subsequent experiments. The expression of activating transcription factor 3 (ATF3) in dorsal root ganglia (DRG) and ionized-calcium-binding adapter molecule-1(Iba-1) in the spinal dorsal horn (SDH) was detected by immunohistochemical staining. In addition, the knee histological assessment was performed by hematoxylin-eosin (HE) staining. RESULTS: The results indicated that injection of MIA induced mechanical allodynia and thermal hyperalgesia, which could be reversed by PRP treatment. PRP downregulated the expression of ATF3 within the DRG and Iba-1 within the SDH. Furthermore, an inhibitory effect on cartilage degeneration was observed in the MIA + PRP group only on day 28. CONCLUSION: These results indicate that PRP intra-articular injection therapy may be a potential therapeutic agent for relieving NP induced by KOA. This effect could be attributed to downregulation of microglial activation and reduction in nerve injury.

Glioblastoma stem cells deliver ABCB4 transcribed by ATF3 via exosomes conferring glioblastoma resistance to temozolomide.

Glioblastoma stem cells (GSCs) play a key role in glioblastoma (GBM) resistance to temozolomide (TMZ) chemotherapy. With the increase in research on the tumour microenvironment, exosomes secreted by GSCs have become a new focus in GBM research. However, the molecular mechanism by which GSCs affect drug resistance in GBM cells via exosomes remains unclear. Using bioinformatics analysis, we identified the specific expression of ABCB4 in GSCs. Subsequently, we established GSC cell lines and used ultracentrifugation to extract secreted exosomes. We conducted in vitro and in vivo investigations to validate the promoting effect of ABCB4 and ABCB4-containing exosomes on TMZ resistance. Finally, to identify the transcription factors regulating the transcription of ABCB4, we performed luciferase assays and chromatin immunoprecipitation-quantitative PCR. Our results indicated that ABCB4 is highly expressed in GSCs. Moreover, high expression of ABCB4 promoted the resistance of GSCs to TMZ. Our study found that GSCs can also transmit their highly expressed ABCB4 to differentiated glioma cells (DGCs) through exosomes, leading to high expression of ABCB4 in these cells and promoting their resistance to TMZ. Mechanistic studies have shown that the overexpression of ABCB4 in GSCs is mediated by the transcription factor ATF3. In conclusion, our results indicate that GSCs can confer resistance to TMZ in GBM by transmitting ABCB4, which is transcribed by ATF3, through exosomes. This mechanism may lead to drug resistance and recurrence of GBM. These findings contribute to a deeper understanding of the mechanisms underlying drug resistance in GBM and provide novel insights into its treatment.

Iridium(III)-Based PD-L1 Agonist Regulates p62 and ATF3 for Enhanced Cancer Immunotherapy.

Anti-PD-L1 immunotherapy, a new lung cancer treatment, is limited to a few patients due to low PD-L1 expression and tumor immunosuppression. To address these challenges, the upregulation of PD-L1 has the potential to elevate the response rate and efficiency of anti-PD-L1 and alleviate the immunosuppression of the tumor microenvironment. Herein, we developed a novel usnic acid-derived Iridium(III) complex, Ir-UA, that boosts PD-L1 expression and converts "cold tumors" to "hot". Subsequently, we administered Ir-UA combined with anti-PD-L1 in mice, which effectively inhibited tumor growth and promoted CD4+ and CD8+ T cell infiltration. To our knowledge, Ir-UA is the first iridium-based complex to stimulate the expression of PD-L1 by explicitly regulating its transcription factors, which not only provides a promising platform for immune checkpoint blockade but, more importantly, provides an effective treatment strategy for patients with low PD-L1 expression.

ATF3/EGR1 regulates myocardial ischemia/reperfusion injury induced autophagy and inflammation in cardiomyocytes.

Myocardial ischemia/reperfusion injury (MIRI) is an irreversible adverse event during the management of coronary heart disease that lacks effective controls. The underlying mechanism of MIRI still requires further investigation. Recent studies have suggested that overexpression of ATF3 protects against MIRI by regulating inflammatory responses, ferroptosis, and autophagy. The downstream target of ATF3, EGR1, also showed cardioprotective properties against MIRI by promoting autophagy. Therefore, further investigating the effect of ATF3/EGR1 pathway on MIRI-induced inflammation and autophagy is needed. Cardiomyocyte MIRI model was established by challenging H9C2 cells with hypoxia/reoxygenation (H/R). The ATF3 overexpression-H/R cell model by transfecting ATF3 plasmid into the H9C2 cell line. The transcription levels of ATF3 and EGR1 were determined using RT-qPCR, the levels of TNF-α and IL-6 were determined using ELISA kits, the protein expression of LC3 I, LC3 II, and P62 was determined via WB, and microstructure of H9C2 cell was observed by transmission electron microscopy (TEM). Overexpression of ATF3 significantly downregulated Egr1 levels, indicating that EGR1 might be the target of ATF3. By upregulating ATF3 levels, the extracellular levels of the inflammatory cytokines TNF-α and IL-6 significantly decreased, and the protein expression of the autophagy markers LC3 I, LC3 II, and P62 significantly increased. TEM results revealed that the cell line in the H/R-ATF3 group exhibited a higher abundance of autophagosome enclosures of mitochondria. The results indicated that ATF3/EGR1 may alleviate inflammation and improve autophagy in an H/R-induced MIRI model of cardiomyocytes.

ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC.

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

MSC-derived mitochondria promote axonal regeneration via Atf3 gene up-regulation by ROS induced DNA double strand breaks at transcription initiation region.

BACKGROUND: The repair of peripheral nerve injury poses a clinical challenge, necessitating further investigation into novel therapeutic approaches. In recent years, bone marrow mesenchymal stromal cell (MSC)-derived mitochondrial transfer has emerged as a promising therapy for cellular injury, with reported applications in central nerve injury. However, its potential therapeutic effect on peripheral nerve injury remains unclear. METHODS: We established a mouse sciatic nerve crush injury model. Mitochondria extracted from MSCs were intraneurally injected into the injured sciatic nerves. Axonal regeneration was observed through whole-mount nerve imaging. The dorsal root ganglions (DRGs) corresponding to the injured nerve were harvested to test the gene expression, reactive oxygen species (ROS) levels, as well as the degree and location of DNA double strand breaks (DSBs). RESULTS: The in vivo experiments showed that the mitochondrial injection therapy effectively promoted axon regeneration in injured sciatic nerves. Four days after injection of fluorescently labeled mitochondria into the injured nerves, fluorescently labeled mitochondria were detected in the corresponding DRGs. RNA-seq and qPCR results showed that the mitochondrial injection therapy enhanced the expression of Atf3 and other regeneration-associated genes in DRG neurons. Knocking down of Atf3 in DRGs by siRNA could diminish the therapeutic effect of mitochondrial injection. Subsequent experiments showed that mitochondrial injection therapy could increase the levels of ROS and DSBs in injury-associated DRG neurons, with this increase being correlated with Atf3 expression. ChIP and Co-IP experiments revealed an elevation of DSB levels within the transcription initiation region of the Atf3 gene following mitochondrial injection therapy, while also demonstrating a spatial proximity between mitochondria-induced DSBs and CTCF binding sites. CONCLUSION: These findings suggest that MSC-derived mitochondria injected into the injured nerves can be retrogradely transferred to DRG neuron somas via axoplasmic transport, and increase the DSBs at the transcription initiation regions of the Atf3 gene through ROS accumulation, which rapidly release the CTCF-mediated topological constraints on chromatin interactions. This process may enhance spatial interactions between the Atf3 promoter and enhancer, ultimately promoting Atf3 expression. The up-regulation of Atf3 induced by mitochondria further promotes the expression of downstream regeneration-associated genes and facilitates axon regeneration.

Revealing the mechanism of ethyl acetate extracts of Semen Impatientis against prostate cancer based on network pharmacology and transcriptomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostate cancer (PCa) is the most common malignancy of the male genitourinary system and currently lacks effective treatment. Semen Impatientis, the dried ripe seed of Impatiens balsamina L., is described by the Chinese Pharmacopoeia as a traditional Chinese medicine (TCM) and is used in clinical practice to treat tumors, abdominal masses, etc. In our previous study, the ethyl acetate extracts of Semen Impatientis (EAESI) was demonstrated to be the most effective extract against PCa among various extracts. However, the biological effects of EAESI against PCa in vivo and the specific antitumor mechanisms involved remain unknown. AIM OF THE STUDY: In this study, we aimed to investigate the antitumor effect of EAESI on PCa in vitro and in vivo by performing network pharmacology analysis, transcriptomic analysis, and experiments to explore and verify the underlying mechanisms involved. MATERIALS AND METHODS: The antitumor effect of EAESI on PCa in vitro and in vivo was investigated via CCK-8, EdU, flow cytometry, and wound healing assays and xenograft tumor models. Network pharmacology analysis and transcriptomic analysis were employed to explore the underlying mechanism of EAESI against PCa. Activating transcription factor 3 (ATF3) and androgen receptor (AR) were confirmed to be the targets of EAESI against PCa by RT‒qPCR, western blotting, and rescue assays. In addition, the interaction between ATF3 and AR was assessed by coimmunoprecipitation, immunofluorescence, and nuclear-cytoplasmic separation assays. RESULTS: EAESI decreased cell viability, inhibited cell proliferation and migration, and induced apoptosis in AR+ and AR- PCa cells. Moreover, EAESI suppressed the growth of xenograft tumors in vivo. Network pharmacology analysis revealed that the hub targets of EAESI against PCa included AR, AKT1, TP53, and CCND1. Transcriptomic analysis indicated that activating transcription factor 3 (ATF3) was the most likely critical target of EAESI. EAESI downregulated AR expression and decreased the transcriptional activity of AR through ATF3 in AR+ PCa cells; and EAESI promoted the expression of ATF3 and exerted its antitumor effect via ATF3 in AR+ and AR- PCa cells. CONCLUSIONS: EAESI exerts good antitumor effects on PCa both in vitro and in vivo, and ATF3 and AR are the critical targets through which EAESI exerts antitumor effects on AR+ and AR- PCa cells.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer.

The induction of ferroptosis is promising for cancer therapy. However, the mechanisms enabling cancer cells to evade ferroptosis, particularly in low-cystine environments, remain elusive. Our study delves into the intricate regulatory mechanisms of Activating transcription factor 3 (ATF3) on Cystathionine ß-synthase (CBS) under cystine deprivation stress, conferring resistance to ferroptosis in colorectal cancer (CRC) cells. Additionally, our findings establish a positively correlation between this signaling axis and CRC progression, suggesting its potential as a therapeutic target. Mechanistically, ATF3 positively regulates CBS to resist ferroptosis under cystine deprivation stress. In contrast, the suppression of CBS sensitizes CRC cells to ferroptosis through targeting the mitochondrial tricarboxylic acid (TCA) cycle. Notably, our study highlights that the ATF3-CBS signaling axis enhances ferroptosis-based CRC cancer therapy. Collectively, the findings reveal that the ATF3-CBS signaling axis is the primary feedback pathway in ferroptosis, and blocking this axis could be a potential therapeutic approach for colorectal cancer.

EPIC-0628 abrogates HOTAIR/EZH2 interaction and enhances the temozolomide efficacy via promoting ATF3 expression and inhibiting DNA damage repair in glioblastoma.

The efficacy of temozolomide (TMZ) treatment in glioblastoma (GBM) is influenced by various mechanisms, mainly including the level of O6-methylguanine-DNA methyltransferase (MGMT) and the activity of DNA damage repair (DDR) pathways. In our previous study, we had proved that long non-coding RNA HOTAIR regulated the GBM progression and mediated DDR by interacting with EZH2, the catalytic subunit of PRC2. In this study, we developed a small-molecule inhibitor called EPIC-0628 that selectively disrupted the HOTAIR-EZH2 interaction and promoted ATF3 expression. The upregulation of ATF3 inhibited the recruitment of p300, p-p65, p-Stat3 and SP1 to the MGMT promoter. Hence, EPIC-0628 silenced MGMT expression. Besides, EPIC-0628 induced cell cycle arrest by increasing the expression of CDKN1A and impaired DNA double-strand break repair via suppressing the ATF3-p38-E2F1 pathway. Lastly, EPIC-0628 enhanced TMZ efficacy in GBM in vitro and vivo. Hence, this study provided evidence for the combination of epigenetic drugs EPIC-0628 with TMZ for GBM treatment through the above mechanisms.

STEAP3 promotes colon cancer cell proliferation and migration via regulating histone acetylation.

Colorectal cancer (CRC) is the third most prevalent diagnosed cancer in men and second most prevalent cancer in women. H3K27ac alterations are more commonly than gene mutations in colorectal cancer. Most colorectal cancer genes have significant H3K27ac changes, which leads to an over-expression disorder in gene transcription. Over-expression of STEAP3 is involved in a variety of tumors, participating in the regulation of cancer cell proliferation and migration. The purpose of this work is to investigate the role of STEAP3 in the regulation of histone modification (H3K27ac) expression in colon cancer. Bioinformatic ChIP-seq, ChIP-qPCR and ATAC-seq were used to analyze the histone modification properties and gene accessibility of STEAP3. Western blot and qRT-PCR were used to evaluate relative protein and gene expression, respectively. CRISPR/Cas9 technology was used to knockout STEAP3 on colon cancer cells to analyze the effect of ATF3 on STEAP3. STEAP3 was over-expressed in colon cancer and associated with higher metastases and more invasive and worse stage of colon cancer. ChIP-seq and ChIP-qPCR analyses revealed significant enrichment of H3K27ac in the STEAP3 gene. In addition, knocking down STEAP3 significantly inhibits colon cancer cell proliferation and migration and down-regulates H3K27ac expression. ChIP-seq found that ATF3 is enriched in the STEAP3 gene and CRISPR/Cas9 technology used for the deletion of the ATF3 binding site suppresses the expression of STEAP3. Over-expression of STEAP3 promotes colon cancer cell proliferation and migration. Mechanical studies have indicated that H3K27ac and ATF3 are significantly enriched in the STEAP3 gene and regulate the over-expression of STEAP3.

FATP2 activates PI3K/Akt/mTOR pathway by inhibiting ATF3 and promotes the occurrence and development of bladder cancer.

Bladder cancer (BLCA) is ranked among the main causes of mortality in male cancer patients, and research into targeted therapies guided by its genomics and molecular biology has been a prominent focus in BLCA studies. Fatty acid transporter protein 2 (FATP2), a member of the FATPs family,is a key contributor to the progression of cancers such as hepatocellular carcinomas and melanomas.However,its role in BLCA remains poorly understand. This study delved into the function of FATP2 in BLCA through a succession of experiments in vivo and in vitro, employing techniques as quantitative real-time polymerase chain reaction (qRT-PCR), RNA sequencing, transwell assays, immunofluorescence, western blot,and others to dissect its mechanistic actions. The findings revealed that an oncogenic function is executed by FATP2 in bladder cancer, significantly impacting the proliferation and migration capabilities, thereby affecting the prognosis of BLCA patients. Furthermore, A suppression that relies on both time and concentration of BLCA proliferation and migration, trigger of apoptosis, and blockage of the cell cycle at the G2/M phase were observed when the inhibitor of FATP2, Lipofermata, was applied. It was unveiled through subsequent investigations that ATF3 expression is indirectly promoted by Lipofermata through the inhibition of FATP2, ultimately inhibiting the signal transduction of the PI3K/Akt/mTOR pathway. This effect was also responsible for the inhibitory impact on BLCA proliferation. Therefore, FATP2 emerges as an auspicious and emerging molecular target with potential applications in precision therapy in BLCA.

Exponential Combination of a and e/g Intracellular Peptide Libraries Identifies a Selective ATF3 Inhibitor.

Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C-C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents ∼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.

Role of ATF3 triggering M2 macrophage polarization to protect against the inflammatory injury of sepsis through ILF3/NEAT1 axis.

BACKGROUND: Sepsis is a systemic inflammatory response which is frequently associated with acute lung injury (ALI). Activating transcription factor 3 (ATF3) promotes M2 polarization, however, the biological effects of ATF3 on macrophage polarization in sepsis remain undefined. METHODS: LPS-stimulated macrophages and a mouse model of cecal ligation and puncture (CLP)-induced sepsis were generated as in vitro and in vivo models, respectively. qRT-PCR and western blot were used to detect the expression of ATF3, ILF3, NEAT1 and other markers. The phenotypes of macrophages were monitored by flow cytometry, and cytokine secretion was measured by ELISA assay. The association between ILF3 and NEAT1 was validated by RIP and RNA pull-down assays. RNA stability assay was employed to assess NEAT1 stability. Bioinformatic analysis, luciferase reporter and ChIP assays were used to study the interaction between ATF3 and ILF3 promoter. Histological changes of lung tissues were assessed by H&E and IHC analysis. Apoptosis in lungs was monitored by TUNEL assay. RESULTS: ATF3 was downregulated, but ILF3 and NEAT1 were upregulated in PBMCs of septic patients, as well as in LPS-stimulated RAW264.7 cells. Overexpression of ATF3 or silencing of ILF3 promoted M2 polarization of RAW264.7 cells via regulating NEAT1. Mechanistically, ILF3 was required for the stabilization of NEAT1 through direct interaction, and ATF3 was a transcriptional repressor of ILF3. ATF3 facilitated M2 polarization in LPS-stimulated macrophages and CLP-induced septic lung injury via ILF3/NEAT1 axis. CONCLUSION: ATF3 triggers M2 macrophage polarization to protect against the inflammatory injury of sepsis through ILF3/NEAT1 axis.

Nucleus pulposus cells regulate macrophages in degenerated intervertebral discs via the integrated stress response-mediated CCL2/7-CCR2 signaling pathway.

Lower back pain (LBP), which is a primary cause of disability, is largely attributed to intervertebral disc degeneration (IDD). Macrophages (MΦs) in degenerated intervertebral discs (IVDs) form a chronic inflammatory microenvironment, but how MΦs are recruited to degenerative segments and transform into a proinflammatory phenotype remains unclear. We evaluated chemokine expression in degenerated nucleus pulposus cells (NPCs) to clarify the role of NPCs in the establishment of an inflammatory microenvironment in IDD and explored the mechanisms. We found that the production of C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 7 (CCL7) was significantly increased in NPCs under inflammatory conditions, and blocking CCL2/7 and their receptor, C-C chemokine receptor type 2(CCR2), inhibited the inductive effects of NPCs on MΦ infiltration and proinflammatory polarization. Moreover, activation of the integrated stress response (ISR) was obvious in IDD, and ISR inhibition reduced the production of CCL2/7 in NPCs. Further investigation revealed that activating Transcription Factor 3 (ATF3) responded to ISR activation, and ChIP-qPCR verified the DNA-binding activity of ATF3 on CCL2/7 promoters. In addition, we found that Toll-like receptor 4 (TLR4) inhibition modulated ISR activation, and TLR4 regulated the accumulation of mitochondrial reactive oxygen species (mtROS) and double-stranded RNA (dsRNA). Downregulating the level of mtROS reduced the amount of dsRNA and ISR activation. Deactivating the ISR or blocking CCL2/7 release alleviated inflammation and the progression of IDD in vivo. Moreover, MΦ infiltration and IDD were inhibited in CCR2-knockout mice. In conclusion, this study highlights the critical role of TLR4/mtROS/dsRNA axis-mediated ISR activation in the production of CCL2/7 and the progression of IDD, which provides promising therapeutic strategies for discogenic LBP.

Co-catalpol alleviates fluoxetine-induced main toxicity: Involvement of ATF3/FSP1 signaling-mediated inhibition of ferroptosis.

BACKGROUND: Fluoxetine is often used as a well-known first-line antidepressant. However, it is accompanied with hepatogenic injury as its main organ toxicity, thereby limiting its application despite its superior efficacy. Fluoxetine is commonly traditionally used combined with some Chinese antidepressant prescriptions containing Rehmannia glutinosa (Dihuang) for depression therapy and hepatoprotection. Our previous experiments showed that co-Dihuang can alleviate fluoxetine-induced liver injury while efficiencies, and catalpol may be the key ingredient to characterize the toxicity-reducing and synergistic effects. However, whether co-catalpol can alleviate fluoxetine-induced liver injury and its toxicity-reducing mechanism remain unclear. PURPOSE: On the basis of the first recognition of the dose and duration at which pre-fluoxetine caused hepatic injury, co-catalpol's alleviation of fluoxetine-induced hepatic injury and its pathway was comprehensively elucidated. METHOD AND RESULTS: The hepatoprotection of co-catalpol was evaluated by serum biochemical indexes sensitive to hepatic injury and multiple staining techniques for hepatic pathologic analysis. Subsequently, the pathway by which catalpol alleviated fluoxetine-induced hepatic injury was predicted by network pharmacology to be predominantly the inhibition of ferroptosis. These were validated and confirmed in subsequent experiments with key technologies and diagnostic reagents related to ferroptosis. Further molecular docking showed that activating transcription factor 3 (ATF3) and ferroptosis suppressor protein 1 (FSP1) were the the most prospective molecules for catalpol and fluoxetine among many ferroptosis-related molecules. The critical role of ATF3/FSP1 signaling was further observed by surface plasmon resonance, diagnostic reagents, transmission electron microscopy, Western blot, real-time PCR, immunofluorescence, and immunohistochemistry. Results showed that fluoxetine directly bound to ATF3 and FSP1; agonisting ATF3 or blocking FSP1 abolished the alleviation of catalpol on fluoxetine-induced liver injury, and both exacerbated ferroptosis. Moreover, co-catalpol significantly enhanced the antidepressant efficacy of fluoxetine against depressive behaviours in mice. CONCLUSION: The hepatic impairment properties of fluoxetine were largely dependent on ATF3/FSP1 target-mediated ferroptosis. Co-catalpol alleviated fluoxetine-induced hepatic injury while enhancing its antidepressant efficacy, and that ATF3/FSP1 signaling-mediated inhibition of ferroptosis was involved in its co-administration detoxification mechanism. This study was the first to reveal the hepatotoxicity characteristics, targets, and mechanisms of fluoxetine; provide a detoxification and efficiency regimen by co-catalpol; and elucidate the detoxification mechanism.

Discovery of a Novel Series of Homo sapiens Caseinolytic Protease P Agonists for Colorectal Adenocarcinoma Treatment via ATF3-Dependent Integrated Stress Response.

Homo sapiens caseinolytic protease P (HsClpP) activation is a promising strategy for colon cancer treatment. In this study, CCG1423 was identified as a selective activator of HsClpP. After optimization, NCA029 emerged as the most potent compound, with an EC50 of 0.2 µM against HsClpP. Molecular dynamics revealed that the affinity of NCA029 for the YYW aromatic network is crucial for its selectivity toward HsClpP. Furthermore, NCA029 displayed favorable pharmacokinetics and safety profiles and significantly inhibited tumor growth in HCT116 xenografts, resulting in 83.6% tumor inhibition. Mechanistically, NCA029 targeted HsClpP, inducing mitochondrial dysfunction and activating the ATF3-dependent integrated stress response, ultimately causing cell death in colorectal adenocarcinoma. These findings highlight NCA029 as an effective HsClpP activator with potential for colon cancer therapy.

SIRT1 activated by AROS sensitizes glioma cells to ferroptosis via induction of NAD+ depletion-dependent activation of ATF3.

Ferroptosis is a type of programmed cell death resulting from iron overload-dependent lipid peroxidation, and could be promoted by activating transcription factor 3 (ATF3). SIRT1 is an enzyme accounting for removing acetylated lysine residues from target proteins by consuming NAD+, but its role remains elusive in ferroptosis and activating ATF3. In this study, we found SIRT1 was activated during the process of RSL3-induced glioma cell ferroptosis. Moreover, the glioma cell death was aggravated by SIRT1 activator SRT2183, but suppressed by SIRT inhibitor EX527 or when SIRT1 was silenced with siRNA. These indicated SIRT1 sensitized glioma cells to ferroptosis. Furthermore, we found SIRT1 promoted RSL3-induced expressional upregulation and nuclear translocation of ATF3. Silence of ATF3 with siRNA attenuated RSL3-induced increases of ferrous iron and lipid peroxidation, downregulation of SLC7A11 and GPX4 and depletion of cysteine and GSH. Thus, SIRT1 promoted glioma cell ferroptosis by inducting ATF3 activation. Mechanistically, ATF3 activation was reinforced when RSL3-induced decline of NAD+ was aggravated by FK866 that could inhibit NAD + synthesis via salvage pathway, but suppressed when intracellular NAD+ was maintained at higher level by supplement of exogenous NAD+. Notably, the NAD + decline caused by RSL3 was enhanced when SIRT1 was further activated by SRT2183, but attenuated when SIRT1 activation was inhibited by EX527. These indicated SIRT1 promoted ATF3 activation via consumption of NAD+. Finally, we found RSL3 activated SIRT1 by inducing reactive oxygen species-dependent upregulation of AROS. Together, our study revealed SIRT1 activated by AROS sensitizes glioma cells to ferroptosis via activation of ATF3-dependent inhibition of SLC7A11 and GPX4.

Activating transcription factor 3 is a new biomarker correlation with renal clear cell carcinoma progression.

Background: Clear cell renal cell carcinoma (ccRCC) is the most invasive type of cancer, with a high risk of metastasis and recurrence. Therefore, there is an urgent need to identify novel prognostic predictors and therapeutic targets of ccRCC. Activating transcription factor 3 (ATF3), a tumor oncogene or repressor, has rarely been examined in ccRCC. In the present study, we comprehensively elucidate the prognostic value and potential functions of ATF3 in ccRCC.Methods: Several TCGA-based online databases were used to analyze ATF3 expression in ccRCC and determine ccRCC prognosis. The upstream-binding micro (mi) RNAs of ATF3 and long non-coding (lnc)RNAs were predicted using the StarBase database.Results: Analysis of several TCGA-based online databases showed that ATF3 expression is decreased in ccRCC, suggesting a significant association with the prognosis of patients with ccRCC. Furthermore, we found hsa-miR-221-3p to be potential regulatory miRNA of ATF3 in ccRCC. Prediction and analysis of the upstream lncRNAs indicated that PAXIP1-AS2 and OIP5-AS1 were the most potent upstream lncRNAs of the hsa-miR-221-3p/ATF3 axis in ccRCC. The results of the GO and KEGG analyses implied that ATF3 is likely involved in the regulation of apoptotic signaling in response to endoplasmic reticulum (ER) stress in ccRCC. Correlation analysis revealed a positive relationship between ATF3 expression and ER stress.Conclusions: Our in silico findings highlighted that ATF3 expression was low in ccRCC and negatively correlated with poor prognosis. Furthermore, PAXIP1-AS2 and the OIP5-AS1/hsa-miR-221-3p/ATF3 axis were identified as significant potential regulators of ER stress-mediated apoptosis in ccRCC.

The Dual Roles of Activating Transcription Factor 3 (ATF3) in Inflammation, Apoptosis, Ferroptosis, and Pathogen Infection Responses.

Transcription factors are pivotal regulators in the cellular life process. Activating transcription factor 3 (ATF3), a member of the ATF/CREB (cAMP response element-binding protein) family, plays a crucial role as cells respond to various stresses and damage. As a transcription factor, ATF3 significantly influences signal transduction regulation, orchestrating a variety of signaling pathways, including apoptosis, ferroptosis, and cellular differentiation. In addition, ATF3 serves as an essential link between inflammation, oxidative stress, and immune responses. This review summarizes the recent advances in research on ATF3 activation and its role in regulating inflammatory responses, cell apoptosis, and ferroptosis while exploring the dual functions of ATF3 in these processes. Additionally, this article discusses the role of ATF3 in diseases related to pathogenic microbial infections. Our review may be helpful to better understand the role of ATF3 in cellular responses and disease progression, thus promoting advancements in clinical treatments for inflammation and oxidative stress-related diseases.